The ultimate goal of this research project is the development of therapeutic agents for diseases that can be treated by inhibiting zinc hydrolytic enzymes, including rheumatoid and osteoarthritis, periodontitis, glaucoma, and cancer. The design of these agents requires a knowledge of (1) the mechanism of the enzyme and (2) the energies of binding interactions between the enzyme and an inhibitor. The catalytic mechanisms and inhibitor binding energies are the sum of many varied forces. It is difficult to measure the contribution of an individual type of interaction in an enzyme due to its size and complexity. This proposal focuses on two of the most highly studied zinc hydrolytic enzymes, carbonic anhydrase and carboxypeptidase A. It is believed that many other zinc enzymes are similar in structure and utilize similar mechanisms. We have chosen to address specifically the means by which enzymes activate water that is coordinated to zinc in the active site. Studies of carbonic anhydrase suggest that hydrophobic interactions, hydrogen bonds, and the presence of carboxylates near the zinc ion or the water affect enzyme activity and inhibitor binding, but the magnitude and the detailed physical basis for the effects are unknown. This proposal seeks to quantify the effects and establish how and why the stated interactions operate. In carboxypeptidase A, the overall mechanistic path is still a matter of controversy, to say nothing of the details of the path. There is presently no ligand that forms a complex with zinc and has ligands and geometry which closely resemble those found in CPA. This proposal contains a plan to develop such a ligand. Binding studies will be used to determine the energy of inhibitor coordination to zinc in the absence of secondary interactions in the enzyme active site. Once this goal has been achieved, ligands will be prepared which quantify the contribution of some of the known active site secondary interactions. Proposed mechanisms of action of carboxypeptidase A will be tested in carefully designed model complexes. The tools used to achieve the goals of this study include molecular design and synthesis of model coordination complexes. The complexes will be characterized by x-ray crystallography, potentiometry, calorimetry, kinetic studies, and other techniques.